Cell-to-cell signaling in bacteria, also known as quorum sensing, involves the production and detection of small molecules, or pheromones, that are exchanged between bacteria. Quorum sensing is used to coordinate important physiological processes such as virulence development, biofilm production, and the ability to transfer genetic information between cells, also known as competence. Genetic disruption of these pathways can severely affect the capability of bacteria to perform those functions. Therefore, defining these signaling mechanisms, and developing methods to interfere with them could provide novel strategies to treat bacterial infections. Streptococcus mutans, the primary agent of dental caries, uses a quorum sensing pathway involving a short peptide pheromone called XIP and its cognate receptor, ComR. XIP develops from a precursor peptide called ComS that is secreted from the cell and processed into the mature pheromone. For XIP to be detected, it is transported into the cytosol where it is bound by ComR. The ComR/XIP complex induces a transcriptional cascade that results in development of the competent state. Interestingly, the ComRS pathway and all known components of the competence system are conserved across all members of the Mutans, Pyogenic and Bovis groups. Our data indicate that although XIP orthologs are generally specific to each species, there is evidence of cross-talk between species and groups of streptococci, We hypothesize that interactions between ComR and XIP of different species are promiscuous. Aim 1 of this proposal seeks to define the specificity and promiscuity between the ComR and ComS proteins of multiple streptococcal species. Using an S. mutans 'test bed' strain that we developed to contain a luciferase reporter monitoring ComR activity , we will test interactions of con- and hetero-specific pairings of XIP and ComR alleles. These tests will allow us to determine whether ComR and ComS proteins from different species are able to successfully interact to induce luciferase activity. By characterizing the interactions between all combinations of ComR and ComS proteins, we will gain evidence as to what is necessary for a successful interaction. Aim 2 of this proposal involves screening genetic libraries of ComS variants to identify peptides that inhibit signaling. We have designed a method to generate large and diverse libraries of ComS-derived peptides. Using the power of bacterial genetics, we will screen for peptide variants that block productive interactions between XIP and ComR. The identification of inhibitors will aid our abilities to define components of XIP that are critical t generate a productive signal, and will also allow us to begin developing methods to interfere with other pheromone-dependent signaling pathways in Gram-positive bacteria.